Binding assay employing labelled reagent

ABSTRACT

A binding assay process for an analyte, using a capture binding agent with binding sites specific for the analyte and a developing binding material capable of binding with the bound analyte or with the binding sites on the capture binding agent either occupied by the bound analyte or the remaining unoccupied binding sites, employs the capture binding agent in an amount such that only an insignificant fraction of the sample analyte becomes bound to the capture binding agent, which is preferably provided at high surface density on microspots. A label is used in relation to the developing binding material and is provided by microspheres which are less than 5 μm and carry a marker preferably fluorescent dye molecules. To determine the concentration of sample analyte, the signal strength, which represents the fractional occupancy of the binding sites on the capture binding agent by the analyte, is compared with a dose-response curve computed from standard samples. To detect an analyte comprising a single-stranded DNA sequence the analyte presence is detected by the existence of a signal. A kit for the process comprises the capture binding agent immobilised on a solid support, a developing reagent with the developing binding material attached to the microspheres and, for quantitative assays, standards of known amounts of concentrations of the analyte of interest.

FIELD OF THE INVENTION

The present invention relates to binding assays employing a labelledreagent. Binding assays include immunoassays for the determination ofconcentrations of antigens in liquid samples, and it is also possible touse the present invention for the determination or detection of otheranalytes in liquid samples, including DNA sequences.

The present invention has particular relevance to non-competitivesandwich assays, that is to say assays in which a liquid samplecontaining an antigen or other analyte to be assayed such as a hormoneis contacted with a first binding agent (such as an antibody) havingbinding sites on its molecule specific for the analyte whereby afraction of the binding sites on the first binding agent representativeof the concentration of the analyte in the liquid sample are occupied bythe analyte. The fractional occupancy of the binding sites is thendetermined by a back-titration technique involving the use of a secondbinding material which is capable of binding with the bound analyte orwith the binding sites occupied by bound analyte but not with unoccupiedbinding sites. Conveniently, the first binding agent will be referred tohereinafter as the capture binding agent and the second binding materialwill be referred to hereinafter as the developing binding material.

Non-competitive assays are to be distinguished from competitive assaysin which the back-titration technique involves the use of a developingbinding material which competes with the analyte for the binding siteson the capture binding agent, for example a labelled version of theanalyte or another material able to bind with the unoccupied bindingsites on the capture binding agent, although the present invention canalso be used in such assays. In each case the extent of binding of thedeveloping binding material is determined by the labelling of thedeveloping binding material (or both that material and the capturebinding agent), for example with a fluorescent label, and comparing thestrength of the signal emitted by the bound labelled product of analytebound to capture binding agent and developing binding material in thecase of the unknown sample with the signal strengths achieved withcorresponding samples of known analyte concentration which togetherprovide a dose-response curve. One type of non-competitive sandwichassay involves the use of a labelled developing binding material and animmobilised capture binding agent which may or may not be labelled.

BACKGROUND ART

It is now well-recognised that non-competitive sandwich immunoassaysgenerally display higher sensitivity than the more conventionalcompetitive immunoassays. The widely accepted explanation for thishigher sensitivity is the use of relatively large amounts both of theimmobilised capture binding agent (usually an antibody located on asolid support) and of the labelled developing binding material (alsooften an antibody). By using large amounts of the antibodies, especiallythe capture antibody, the rate of reaction between analyte and captureantibody is increased, implying in accordance with the law of massaction that a greater amount of analyte is captured on the solid phasecapture antibody in any specified time interval. Thus, the use of largeamounts of capture antibody is generally perceived as crucial to thedevelopment of non-competitive immunoassays combining very highsensitivity with relatively short incubation times. (See for example Hayet al. "American Thyroid Association Assessment of Current Free ThyroidHormone and Thyrotropin Measurements and Guidelines for Future ClinicalAssays" in Clinical Chemistry, Vol 37, No. 11, (1991) at pages2002-2008.) This approach nevertheless carries disadvantages. Forexample, it implies heavy consumption of antibodies which may be scarceand costly to produce. It also involves the use of various stratagems tomaximise the total surface area of the solid support on which thecapture antibody is deposited. For example, porous glass microsphereshave been used as a solid support in sandwich assay systems, the poresgreatly increasing the surface area available for antibody attachment.

Roger Ekins has previously argued, for example in WO-84/01031,WO-88/01058 and WO-89/01157, that this general perception is mistakenand that, in certain circumstances, assays which have an even greatersensitivity than that attainable under the conditions mentioned abovecan be developed when the unknown sample and standard samples containingthe analyte are each contacted with such a small amount of the capturebinding agent that only an insignificant fraction of the analyte becomesbound to the capture binding agent. (This insignificant fraction isusually less than 5% and ideally 1-2% or less of the total amount of theanalyte in the sample, bearing in mind that errors in analytedetermination unavoidably introduced into the measuring procedureelsewhere by limitations in the accuracy of sample and reagentmanipulation, signal measurements, standardisation, temperaturevariation and the like are generally of the order of 10% of the analytein the sample, although sometimes the binding of higher fractions of theanalyte up to 10% or so may be tolerated when exact determination isless important.) Only when an insignificant fraction of the total amountof analyte becomes bound is the fractional occupancy F of the bindingsites on the capture binding agent related to the concentration [A] ofanalyte in the sample (at thermodynamic equilibrium) by the equation##EQU1## where K is the affinity constant of the capture binding agentfor the analyte measured at equilibrium and is a constant at a giventemperature and other given conditions. Before thermodynamic equilibriumis reached, the above equation also approximately applies (provided thatonly an insignificant fraction of the analyte in the sample has becomebound to the capture binding agent at the time of measurement of thefractional occupancy, irrespective of whether a higher, significantamount becomes bound subsequently, for example by the time equilibriumis reached), subject only to the alteration that in such a situation theconstant K in the equation is the apparent affinity constant of thecapture binding agent for the analyte at the time of measurement.

It has also been proposed by Roger Ekins in WO-89/01157 etc to carry outsuch a technique using the capture binding agent spotted onto a solidsupport in the form of one or more microspots, for example withdiameters of 1 mm² or less, using sample volumes of the order of 1 ml orless.

However, with such a system a problem may arise to provide a label whichcan give a sufficiently strong but sensitive signal. Doubts have alsobeen expressed regarding sensitivities attainable using microspot assayformats on the ground that the use of very small amounts of solid-phasecapture binding agent must intrinsically necessitate long incubationtimes and yield low sensitivity assays.

SUMMARY OF THE INVENTION

We have now found that in such a system very good results can beobtained by using as the labelling system micron or preferablysub-micron sized microspheres carrying a marker, preferably afluorescent label. By combining the use of such a label for thedeveloping binding material alone or for both the developing bindingmaterial and the capture binding agent with very small amounts ofcapture binding agent located at a high surface density on a solidsupport in the form of a microspot, non-competitive assay systems may bedevised which are as rapid to perform as or even more rapid to performthan, and possess sensitivities comparable with or indeed greatlysuperior to, those of conventional sandwich systems relying uponcomparatively large amounts of capture binding agent. This crucialfinding, which contradicts currently accepted views on the design ofhigh sensitivity assays and is totally unexpected, potentially forms thebasis of development of a variety of superior miniaturized diagnosticdevices possessing exceedingly high sensitivity whilst requiring onlyrelatively short incubation and measurement times.

Of course, the microspheres can also be used for labelling purposes in acompetitive assay system using similarly very small amounts of capturebinding agent, but in such systems the limit on sensitivity may not inpractice be the specific activity of the label, and corresponding orsubstantial increases in sensitivity due to the use of the microsphereswould therefore not necessarily be achieved or even expected, althoughincreases in rapidity can be expected.

According to the present invention there is provided a binding assayprocess in which the concentration of an analyte in a liquid sample isdetermined by comparison with a dose-response curve computed fromstandard samples, using a capture binding agent having binding sitesspecific for the analyte and a developing binding material capable ofbinding with the bound analyte or with the binding sites on the capturebinding agent occupied by the bound analyte or with the binding sitesremaining unoccupied on the capture binding agent, the capture bindingagent being used in an amount such that only an insignificant fractionof the analyte in the sample becomes bound to the capture binding agent,and a label being used in the assay in relation to the developingbinding material whereby the strength of the signal associated with thelabel is representative of the fractional occupancy of the binding siteson the capture binding agent by the analyte, in which process there isused as the label microspheres having a size of less than 5 μm andcarrying a marker, preferably a fluorescent label.

In other embodiments (described below) the present invention provides akit for use in a binding assay process, also a binding assay process forthe detection or determination of an analyte comprising asingle-stranded DNA sequence in a liquid sample.

DETAILED DISCLOSURE

Fluorescent microspheres of micron and submicron size have been knownsince about 1982 and are commercially available from many sources, e.g.from Seradyn Inc. or under the trade mark FluoSpheres from MolecularProbes Inc. Suitable microspheres have a diameter of generally less than5 μm and preferably not more than 1 μm, more preferably of the order of0.01 to 0.5 μm, and it is preferred to use spheres all essentially ofthe same standard size. The microspheres may be made of any suitable orconvenient inert material such as a polymer latex, for example apolystyrene latex, which is desirably provided on its surface witheither negatively charged groups such as sulphate, carboxyl orcarboxylate-modified groups or positively charged groups such as amidinegroups. The presence of such charged groups on the surface of thespheres allows a wide variety of proteins, such as IgG,avidin/streptavidin and BSA, to be adsorbed passively on or coupledcovalently to the surfaces of the spheres at various surface densitiesas desired.

Although the microspheres may carry markers of various types, forexample radioactive, chemiluminescent or enzyme labels, they preferablycarry fluorescent labels. The fluorescent and radioactive labels arepreferably contained within the microspheres, where they are shieldedfrom outside influences, but they may (and the enzyme andchemilumienescent labels will in general) be present on the surface ofthe spheres. Each microsphere desirably contains large numbers offluorescent dye molecules as labels, for example up to 10 million in 1μm diameter spheres with smaller numbers in smaller spheres (e.g. 100 or1,000 to 100,000 or 1 million) down to about 10 in very small spheres.The fluorescent dye molecules may be selected to provide fluorescence ofthe appropriate colour range (excitation and emission wavelength)compatible with standard filter sets, for example yellow/green, orangeor red, or customised filter sets may be used. Fluorescent dyes includecoumarin, fluorescein, rhodamine and Texas Red. The fluorescent dyemolecules may be ones having a prolonged fluorescent period such thatthe strength of the signal emitted by them can be determined by theknown time-resolved fluorescence technique after background interferencehas decayed, for example lanthanide chelates and cryptates. Dyes whichfluoresce only in non-aqueous media can be used. Preferred fluorescentdyes for use in the microspheres are oil-soluble dyes in order tofacilitate their incorporation into the interior of the microspheres.Yellow/green, orange and red FluoSpheres, which are excited veryefficiently at the 488, 568 and 647 nm krypton/argon mixed gas laserlines, respectively, are presently preferred.

In use as the label for the developing binding material, or for thecapture binding agent and the developing binding material, in the assaysystems of the invention the microspheres may have the developingbinding material, or avidin which can be used as a "universal marker"reagent and bind all biotinylated binding material, or the capturebinding agent as the case may be, physically adsorbed onto the surfaceof the spheres. More conveniently, however, appropriatelysurface-modified microspheres are selected and the developing bindingmaterial (eg. antibody) or capture binding agent (eg. antibody) iscovalently bonded to them either directly or through a linking grouping,such as is provided by carbodiimide activation. Thus, for example, tolink the microspheres and binding material the binding material may beadsorbed onto hydrophobic sulphate-modified microspheres or covalentlycoupled to aldehyde-modified or carboxylate-modified hydrophilicmicrospheres, the latter via a water-soluble carbodiimide. When both thecapture binding agent and the developing binding material are labelledwith fluorescent microspheres, different dyes will of course be used inthe two sets of microspheres, and the signal strength ratio can then bedetermined and employed for comparison as in WO 88/01058.

It will be apparent therefore that the microspheres containing manymolecules of a fluorescent dye provide an amplification system (as domicrospheres containing or carrying several molecules of a label ofother types, e.g. a radioactive or chemiluminescent label) in the sensethat one molecule or unit of the developing binding material gives riseto a signal which is due to a large number of fluorescent dye moleculesor a significant number of other label molecules. Such an amplificationsystem is thus able greatly to increase the sensitivity of these assayprocedures in which the controlling factor in assay sensitivity issignal magnitude, for example when only a very small amount of capturebinding agent is used and hence the amount of developing bindingmaterial is also very small.

The microspheres are primarily used in conjunction with an assay systemin which the immobilised material, usually the capture binding agent(capture antibody), is deposited on a solid support in the form of oneor more microspots having an area of 1 mm² down to 100 μm² or less, forexample a diameter of 0.01-1 mm, although for very small microspots itmay be necessary to use very small microspheres or fewer largermicrospheres. The surface density of the capture binding agent on themicrospot is desirably in the range 1,000 to 100,000 molecules/μm²,preferably 10,000 to 50,000 molecules/μm² in the case of antibodies. Forother binding agents the surface density may be within this range orabove or below it but should preferably be as high as possible withoutsterically hindering binding of the analyte molecules. These microspotsare used in conjunction with sample sizes of generally 1 ml or less, forexample down to 50 or 100 μl or even less depending on the size of themicrospot, the aim being to cover the microspot.

The microspot technique can be used to determine different analytes inthe same or different liquid samples in a single operation byimmobilising different capture binding agents on different microspots,for example 10 or more, e.g. up to 50 or more, on the same solid supportand using different or identical developing binding materials labelledwith the microspheres for the different binding assays. The labels (e.g.fluorescent dyes) associated with different binding assays and/or thetechniques used to measure the signal strengths will be chosen to enablethe results from the different assays to be differentiated. Techniquesfor this are known, for example from WO-88/01058.

To optimise the results achievable with the present invention a numberof different features should be optimised, including the following:

i) the fractional occupancy of the capture binding agent by the analyte,

ii) the size of the affinity constant of the capture binding agent forthe analyte at the time when measurement occurs and, if the measurementis to be performed before equilibrium has been reached, the rate atwhich equilibrium is reached,

iii) the surface density of the capture binding agent on its support,

iv) the size of the microspots,

v) the nature of the support,

vi) the instrument used to measure the signal,

vii) the treatment of the microspheres, for example to block unreactedsites, and

viii) the nature of the buffer solutions used.

For feature i) it should be noted that the use of too much capturebinding agent is to be avoided for optimal sensitivity. It istheoretically demonstrable than the highest signal/noise ratio (R) isobtained (assuming the measuring instrument itself generates zero noise)when the amount of capture binding agent falls below 0.01/K andapproaches zero, K being the affinity constant between the capturebinding agent and the analyte at the time of measurement. Let us definethis signal/noise ratio as R_(o). (Note that an amount of capturebinding agent of 0.01/K binds <1% of analyte molecules present in thesolution to which the capture binding agent is exposed.) If the area onwhich the capture binding agent is deposited is increased (the surfacedensity of binding agent remaining constant) the amount of binding agentwill concomitantly increase. The percentage of total analyte bound willalso increase (albeit to a lesser proportional extent) but thesignal/noise ratio will decrease. For example, if the area is increased100-fold so that the amount of binding agent equals 1/K, the amount ofanalyte bound will rise to ≦ 50%, and the signal/noise ratio will fallto the order of R_(o) /2.

The relationship between the ratio R and capture binding agentconcentration (ie area coated with binding agent) is shown in FIG. 1 ofthe accompanying drawings. This Figure is a graph of the signal/noiseratio (the continuous line where the y-axis is % of the value when thearea coated with binding agent, and hence the binding agentconcentration, approaches zero) and the amount of bound analyte (thedashed line, where the y-axis is % of total analyte present in themedium, assuming the total amount of analyte is very low, ie <0.0001/K)as functions of the total amount of capture binding agent (in units of1/K, the x-axis) on the coated area. Clearly, as the area coated withcapture binding agent (and hence its concentration) increases, thepercentage of total analyte bound increases, but the signal/noise ratiofalls. This effect is shown pictorially in FIG. 2 of the accompanyingdrawings, where d is the diameter in mm of the area coated with capturebinding agent, [Ab] is the concentration of binding agent assuming asurface density of 0.1/K per mm², and s/n is the signal/noise ratioexpressed as a percentage of the value observed as the surface areaapproaches zero. This Figure likewise endeavours to show that, as thecoated area increases, the amount of analyte bound also increases butthe signal/noise ratio and hence the sensitivity fall.

However, although the signal/noise ratio R is highest when the bindingagent concentration is less than 0.01/K, it is clear that the ratio maystill be acceptably high when the amount of capture binding agent usedequals or even exceeds 1/K. The upper limit to the amount of bindingagent coated on the microspot area is preferably 10/K. This implies aten-fold lower sensitivity than is achievable using a 1000-fold loweramount of binding agent and it should be emphasized that, although theinvention is capable of yielding very high sensitivity, it is alsoapplicable where lower sensitivity than the maximum attainable isacceptable.

For factor ii) it should be noted that, although at first sight it mightappear to be better to use a capture binding agent with a lowequilibrium value of K, it is in fact generally found that forhigh-sensitivity assays it is better to use binding agents having highervalues of K at equilibrium, e.g. 10¹¹ -10¹² or more liters/mole, and (ifnecessary or desired) to make the measurement before equilibrium hasbeen reached so that at the moment of measurement only an insignificantfraction of the analyte has become bound and the effective value of K atthe moment of measurement may be considerably less, e.g. 10⁸ -10¹¹, eventhough a substantial fraction of the analyte might become bound ifmeasurement were to be delayed until equilibrium had been reached.Higher effective values of K may be more appropriate where the analyteconcentration is low (e.g. 10⁶ molecules/ml) and lower effective valuesof K may be more appropriate where the analyte concentration is high(e.g. 10¹⁴ molecules/ml). Desirably, the effective value of K is of theorder of the reciprocal of the analyte concentration to be measured inthe sample. It is preferred to use a capture binding agent such that, inthe amounts used, equilibrium is reached within 12 hours or somewhatless but to make the measurement within about 2 hours or less, wellbefore equilibrium is reached. This early pre-equilibrium measurement isparticularly important where the capture binding agent has a very highaffinity constant for the analyte, as is the case with DNA probes.

For factor iii) it should be noted that too low a surface densitydecreases the signal/noise ratio because the area occupied by thecapture binding agent and scanned to determine the ratio increases. Onthe other hand too high a surface density can cause steric hindrancebetween adjacent capture binding agent molecules so that not all themolecules are available for binding the analyte. Preliminary experimentsto see if steric hindrance is a problem can be carried out by makingspots of varying binding agent surface density, labelling them with alabel such as 125_(I) and measuring how the signal varies with bindingagent surface density, the optimum being the highest signal. In thepast, conventional practice has been to use coatings of the order of 10μg of capture binding agent (antibody) per ml but for the presentinvention figures of 100-200 μg of capture binding agent (antibody) perml may be more appropriate. The use of higher surface density also hasthe advantage that less of the surface is available for non-specificbinding, which would otherwise increase the noise and reduce thesignal/noise ratio.

For factor v) it should be noted that the support used will itselfcontribute to the noise level. If the level of background noise is aproblem it may be preferable to use a black support rather than a whiteone, although this may decrease the signal level and some black supportshave higher noise levels than others.

For factor vi) it is preferred to maximise the signal/noise ratio.Accordingly, the area from which the signal is measured is desirablysmall, preferably limited to the area of the microspot or a portion ofit. Measuring the signal from a wider area beyond the microspotincreases the noise level without increasing the signal level and thusdecreases the signal/noise ratio. Hence it may be desirable toconcentrate the illumination and to make measurements by means of aconfocal microscope or other instrument achieving very preciseillumination.

For factor vii) it is desirable that, after adsorption or covalentbonding of the developing binding material or capture binding agent tothe microspheres has been carried out, the unreacted sites on themicrospheres are blocked to avoid their non-specific binding to otherbiological molecules or the solid support for the capture binding agent.Blocking may be carried out with any non-interfering protein material.An albumin, particularly bovine serum albumin, is preferred. It has beenfound desirable to block not only with bovine serum albumin (BSA) orequivalent but also with a detergent such as TWEEN-20 or other non-ionicdetergent. It is believed that there are some binding sites on themicrospheres which are not blocked by BSA or other protein materialalone. Microspheres blocked with BSA alone appear still to have bindingsites which are capable of binding to the solid support, such as theplastic walls of the microtitre wells in which the assay is performed,or to other biological or non-biological molecules such as are presentin other components of the system (eg the liquid sample), as well asbeing capable of non-specific binding to the capture binding agent. Useof a detergent as additional blocking agent decreases the number of suchbinding sites or eliminates them altogether. The detergent also helps toremove loosely bound binding agent or material or other proteins whichmight desorb into storage buffer and/or assay buffer and interfere withassays. Non-interfering reactants can be used to block activated groupson the surface of the microspheres, for example inert amines such asethanolamine, glycine or lysine to block activated carboxyl groups, butany particular compounds should be checked for assay compatibility.

For factor viii) it should be noted that the choice of ingredients forthe assay buffer and the wash buffer can influence the sensitivity ofthe results. With TSH, for example, TRIS gives a better buffer thanphosphate. It may also be desirable to include a detergent in the buffersuch as TWEEN 40 to reduce non-specific binding.

In other respects the immunoassay may be carried out in a known manner,for example as described in Roger Ekins' earlier patent applications asmentioned above, and incorporated herein by reference, or in the otherliterature. When carrying out immunoassays it is preferred, although notessential, for both the capture binding agent and the developing bindingmaterial to be antibodies. Monoclonal or polyclonal antibodies may beused and the procedure may be used to assay analytes such as hormones,nucleic acid, proteins, vitamins, drugs, viruses, bacteria, pesticides,rumour markers or other components of biological samples such as bodyfluids, the capture binding agent and developing binding material beingappropriately chosen so as to bind to the analyte in question. Theanalyte in a binding assay can be a nucleotide sequence, eg a DNAoligonucleotide sequence in which case the capture binding agent and thedeveloping binding material may both be other nucleotide sequences,which will differ from one another. The analyte may contain only oneepitope for the capture binding agent or the epitope may be replicatedon the analyte molecule. The polyclonal developing binding material(antibody) may react with a variety of epitopes on the analyte or theanalyte capture binding agent complex, or a mixture of two or moremonoclonal developing binding materials (antibodies) reacting withdifferent epitopes may be used.

When used for nucleic acid (DNA) assays the DNA probe, a single-strandednucleotide sequence, eg an oligonucleotide sequence of conventional orstandard type, is attached as capture binding agent to a solid supportand this recognises a corresponding single-standard DNA sequenceconstituting the analyte in a liquid sample and such sequences becomebound so as to form a twin-stranded sequence. DNA probes consisting ofoligonucleotide sequences are available commercially from a number ofcompanies, e.g. Clontech Laboratories Inc., or they can be synthesisedto order and/or modified (e.g. with biotin or digoxigenin) by commercialcompanies, e.g. British Biotechnology Products Ltd. The developingbinding material may be either a labelled antibody which recognises thetwin-stranded sequence as opposed to single-stranded sequences (see FIG.3 of the accompanying drawings) or another DNA sequence which recognisesanother part of the DNA sequence constituting the analyte and islabelled (see FIG. 4 of the accompanying drawings), both these bindingmaterials producing non-competitive assays. For competitive assays it ispossible to use a labelled developing binding material recognisingunoccupied sites of the capture binding agent, ie residual DNA probe notbound to analyte (see FIG. 5 of the accompanying drawings). In each casethe label is provided in accordance with the invention by themicrospheres carrying a marker, preferably molecules of a fluorescentdye contained within the microspheres. In each of FIGS. 3-5 A representsa microspot, B the capture binding agent and M the microspheres. In FIG.3 AbDSD is an antibody to double stranded DNA, and in FIG. 4 AbD isanti-digoxigenin antibody and D is digoxigenin. Hybridisation techniquesusing these methodologies are already known, see for example:- GuesdonJ-L (1992), "Immunoenzymatic Techniques Applied to the SpecificDetection of Nucleic Acids", Journal of Immunological Methods 150,33-49; Mantero G, Zonaro A, Albertini A, Bertolo P & Primi D. (1991),"DNA Enzyme Immunoassay: General Method for Detecting Products ofPolymerase Chain Reaction", Clinical Chemistry 37/3, 422-429; Keller G.H., Huang D-P, Shih W-K & Manak M. M. (1990), "Detection of Hepatitis BVirus DNA in Serum by Polymerase Chain Reaction Amplification andMicrotiter Sandwich Hybridization", Journal of Clinical Microbiology28/6, 1411-1416; Nickerson D. A., Kaiser R., Lappin S, Stewart J, Hood L& Landegren U (1990), "Automated DNA Diagnostics Using an ELISA-basedOligonucleotide Ligation Assay", Proceedings of the National Academy ofSciences 87, 8923-8927; Wolf S. F., Haines L., Fisch J., Kremsky J. N.,Dougherty J. P. & Jacobs K. (1987), "Rapid Hybridization Kinetics of DNAAttached to Submicron Latex Particles", Nucleic Acids Research 15/7,2911-2926.

DNA assays according to the present invention therefore provide analternative to the well-known polymerase chain reaction (PCR) forassaying DNA sequences. The PCR method is subject to certaindisadvantages, including errors introduced by repeated cycles ofamplification (doubling) on an initial very low concentration of DNAsequence. The present invention provides an alternative enhancementprocedure in which an initial very low concentration of the DNA sequenceto be detected or determined gives rise to an amplified signal in asingle step because of the large number of fluorescent dye moleculescontained within the microsphere to which a molecule of the developingbinding material (antibody or other DNA sequence) is attached as byadsorption or direct or indirect chemical bonding.

According to a further embodiment of the invention there is provided abinding assay process for the detection of an analyte comprisingsingle-stranded DNA sequence in a liquid sample, the process comprisingcontacting the sample in a non-competitive or competitive procedure withan immobilised capture binding agent which is a single-strandedoligonucleotide DNA probe capable of recognising analyte in the liquidsample and binding therewith, and with a labelled developing bindingmaterial which either is an antibody capable of recognising onlytwin-stranded DNA sequences formed from the probe and the analyte and ofbinding therewith or is an oligonucleotide DNA sequence capable ofrecognising and binding with either another part of the analyte or theresidual probe, the developing binding material being labelled by meansof microspheres having a size of less than 5 μm and carrying a marker,and, after the removal of unattached developing binding material,detecting the presence of the analyte by the existence or strength of asignal from the marker attached to developing binding material which hasbecome bonded directly or indirectly to the immobilised capture bindingagent.

Preferably, the marker is a fluorescent label, eg in the form of a largenumber (100 or more) of fluorescent dye molecules contained withinmicrospheres having a size of 0.01 to 1 μm, eg 0.05-0.5 μm. It ispreferred to use this technique in conjunction with the microspottechnique already referred to, with the capture binding agent beingimmobilised as one or more microspots on a solid support at the surfacedensities and microspot sizes already mentioned and optionally differentcapture binding agents being immobilised on different microspots on thesame support to enable a plurality of different DNA sequences to bedetected or determined in a single operation using appropriatelydifferentiated developing binding materials and signal detection orsignal strength measurement techniques.

The procedures for forming a single-stranded DNA probe and immobilisingit on a solid support are well known and described in the literature,for example the Guesdon and other references mentioned above, andstandard techniques can be used for this and for the formation, e.g. byboiling, of the liquid sample containing the analyte (single-strandedDNA sequence which may or may not be present) to be detected. Couplingof the developing binding material to the microspheres may be carriedout by a method known for immobilising to marker-free solid support, forexample as described in the Wolf et al reference mentioned above, andthe usual precautions to avoid contamination etc and other disturbinginfluences should be taken.

In a further embodiment the present invention provides a kit for use ina binding assay process in which the concentration of an analyte in aliquid sample is determined using a capture binding agent having bindingsites specific for the analyte and a developing binding material capableof binding with the bound analyte or with the binding sites on thecapture binding agent occupied by the bound analyte or with the bindingsites remaining unoccupied on the capture binding agent, a label beingused in relation to the developing binding material whereby the strengthof the signal associated with the label is representative of thefractional occupancy of the binding sites on the capture binding agentby the analyte, the kit comprising (a) a solid support having thecapture binding agent immobilised thereon, (b) a developing reagentcomprising the developing binding material adsorbed or directly orindirectly chemically bonded to the surface of microspheres carrying amarker and (c) standards having known amounts or concentrations of theanalyte to be determined.

Preferably, the developing reagent comprises a buffered solutioncontaining the developing binding material attached to the microspheres,but it is also possible to provide the reagent in freeze-dried form.Similarly, the standards may also be provided as buffered solutionscontaining the analyte at known concentrations or in freeze dried formwith instructions for appropriate reconstitution in solution form. Thestandards may be made up in hormone-free serum. There may be three ormore standards, e.g. up to 12, of varying known analyte concentrationsspanning the expected values in the unknown samples.

Preferably, the developing reagent contains the developing bindingmaterial adsorbed onto or covalently bonded to microspheres having asize of less than 5 μm and containing molecules of a fluorescent dye,and it is preferred that the solid support has the capture binding agentimmobilised thereon in the form of one or more microspots of size lessthan 1 mm² and surface density at least 1000 molecules/μm². Differentcapture binding agents may be immobilised on different microspots on thesame solid support and a plurality of different developing reagents anddifferent sets of standards may be provided so that a variety ofdifferent assays for different analytes may be performed using the samesolid support in a single operation, simultaneously or sequentially.

The invention is further described in the following Examples, whichillustrate the preparation of the labelled developing binding material(Examples 1-4) and processes and kits according to the invention(Examples 5-12).

In the Examples concentration percentages are by weight.

EXAMPLE 1

Adsorption of Antibody or Avidin on Hydrophobic Sulfate-Microspheres

1) 0.5 ml of 2% solids suspension in pure water of surfactant-freesulphate-activated microspheres of polymer latex materials containingfluorescent dye molecules within the microspheres (FluoSpheres fromMolecular Probes Inc--FluoSpheres is a Registered Trade Mark) having adiameter 0.08 or 0.12 μm was added dropwise to 2 mg of developingbinding material (antibody or avidin) dissolved in 1 ml of 0.1Mphosphate buffer at pH 7.4. The suspension was shaken overnight at 4° C.

2) The suspension was centrifuged at 20,000 rpm for 30 min at 10° C.(the time and speed of the centrifugation will vary with the size of thelatex microspheres) to separate antibody-conjugated latex microspheresfrom unreacted antibody. The supernatant antibody or avidin solution wasrecovered for protein estimation.

3) The centrifuged pellet was dispersed in 1.0 ml of 0.1M phosphatebuffer by sonication. After dispersion, the unoccupied hydrophobic siteson the microspheres were blocked by the addition of 1 ml of 2% (1%final) bovine serum albumin (BSA) and shaken for 2 hours at roomtemperature. The spheres were further blocked by the addition of 200 μlof 5% Tween-20 (˜0.5% final) and shaken for 1 hour at room temperature.The detergent incubation step also served to get rid of loosely boundantibody/avidin which might desorb into storage and/or assay buffer andwould subsequently interfere with assays.

4) The preparation was centrifuged as above and the microspheresresuspended in 2 ml of 0.1M phosphate buffer.

5) Step 4 was repeated twice. After the final centrifugation, themicrospheres were dispersed in 2 ml of phosphate buffer containing 0.2%BSA and 0.01% sodium azide and stored at 4° C.

EXAMPLE 2

Covalent Coupling of Antibody or Avidin to Carboxylate-Modified LatexMicrospheres by a one-step method

1) 0.5 ml of a 2% solids suspension in pure material ofcarboxylate-modified polymer latex microspheres containing fluorescentdye molecules (FluoSpheres from Molecular Probes Inc) and having adiameter of 0.09 μm was added dropwise to 0.5 ml of 0.015M, pH 5 acetatebuffer containing 2 mg of antibody or avidin as developing bindingmaterial. The suspension was incubated at room temperature for 15 min.

2) 4 mg of EDAC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide] (SigmaChemical Company) was added to the mixture and vortexed. The pH wasadjusted to 6.5±0.2 with dilute NaOH (agglomeration of the latexmicrospheres may be observed at this stage, but they can be redispersedby gentle sonication) and the reaction mixture was mixed gentlyovernight at 4° C.

3) The reaction mixture was centrifuged at 20,000 rpm for 30 min at 10°C. The supernatant was recovered for protein estimation.

4) The centrifuged pellet was dispersed in 1.0 ml of 0.1M phosphatebuffer by sonication. After dispersion, the unoccupied sites on themicrospheres were blocked by the addition of 1 ml of 2% (1% final)bovine serum albumin (BSA) and shaken for 2 hours at room temperature.The spheres were further blocked by the addition of 200 μl of 5%Tween-20 (˜0.5% final) and shaken for a further 1 hour at roomtemperature.

5) The preparation was centrifuged as above and the microspheresresuspended in 2 ml of 0.1M phosphate buffer.

6) Step 5 was repeated twice and, after the final centrifugation, theantibody- or avidin- conjugated microspheres were dispersed in 2 ml ofphosphate buffer containing 0.2% BSA and 0.01% of sodium azide and keptat 4° C.

EXAMPLE 3

Covalent Coupling of an Antibody or Avidin to Carboxylate-Modified LatexMicrospheres by a two-step method

1) 0.5 ml of the suspension of carboxylate-modified latex microspheresused in Example 2 was added to a 10 ml centrifuge tube and centrifugedat 20,000 rpm for 30 min at 10° C.

2) The centrifuged pellet was resuspended in 0.5 ml of 0.02M phosphatebuffer, pH 4.5, and centrifuged as above.

3) Step 2 was repeated.

4) 0.5 ml of a 2% solution of EDAC was added dropwise to the dispersedmicrospheres (agglomeration of the latex microspheres may be observed atthis stage, but they can be redispersed by gentle sonication), and thereaction mixture was mixed gently at room temperature for 3 hours andcentrifuged as above.

5) The centrifuged pellet was resuspended in 1 ml of 0.2M borate buffer,pH 8.5, and centrifuged as above.

6) Step 5 was repeated twice.

7) The centrifuged pellet was resuspended in 0.5 ml of borate buffer,added dropwise to 2 mg of antibody or avidin dissolved in 0.5 ml of thesame buffer and mixed gently overnight at room temperature.

8) The suspension was centrifuged as above and the supernatant was keptfor protein estimation.

9) The centrifuged pellet was resuspended in 1 ml of 0.1M ethanolaminein borate buffer, mixed gently for 30 min at room temperature andcentrifuged as above.

10) The centrifuged pellet was resuspended in 1 ml of 1% BSA, mixedgently for 1 hour and centrifuged as above.

11) The centrifuged pellet was resuspended in 1 ml of 0.5% Tween-20,mixed gently for 1 hour and centrifuged as above.

12) The centrifuged pellet was resuspended in 1 ml of 0.02M phosphatebuffer, pH 7.4, and centrifuged as above.

13) The centrifuged pellet was resuspended in 1 ml of phosphate buffercontaining 0.2% BSA and 0.01% of sodium azide and kept at 4° C.

EXAMPLE 4a

Coupling of a Mixture of Antibody and Avidin to Microspheres byAdsorption or Covalent Linkage

The methodologies for the coupling of a mixture of antibody and avidinto microspheres by adsorption or covalent linkage were essentially thesame as those described in Examples 1 to 3 above for the coupling ofantibody or avidin to microspheres except that the antibody solutionused for the reaction also contained a small amount of avidin.

EXAMPLE 4b

Labelling of a Monoclonal Anti-TSH Antibody with Texas Red

1) 1 mg of monoclonal anti-TSH antibody was dissolved in 1 ml ofcarbonate buffer pH 9.

2) 1 mg of Texas Red (Molecular Probes Inc.) was dissolved in 250 μl ofN,N-Dimethylformamide (Sigma Chemical Company), yielding a concentrationof 4 μg/μl.

3) 10 μl of the 4 μg/μl Texas Red was added to the antibody solution,vortexed and left on ice for two hours (dye to protein ratio(w/w)=0.04).

4) The Texas Red-conjugated antibody was separated from unreacted andhydrolysed dye on a PD10 Sephadex column (Pharmacia) by elution with0.1M phosphate buffer, pH 7.4.

5) Sodium azide was added to the labelled antibody (0.1%) aspreservative and the preparation was stored at 4° C.

EXAMPLE 4c

Labelling of Antibody or BSA with Biotin

1) 2 mg of antibody or BSA was dissolved in 1 ml of pH 8.5 bicarbonatebuffer.

2) 2.2 mg of N-Hydroxysuccinimidyl 6-(Biotinamido) Hexanoate (VectorLaboratories) was dissolved in 55 μl of N,N-Dimethylformamide, yieldinga concentration of 40 μg/μl.

3) 10 μl of the 40 μg/μl biotin was added to the antibody or BSAsolution and shaken for 2 hours at room temperature (biotin to proteinratio (w/w)=0.2).

4) The reaction was terminated by the addition of 10 mg of glycine.

5) The biotin-conjugated IgG or BSA was separated from unreacted biotinon a PD10 Sephadex column (Pharmacia) by elution with 0.1M phosphatebuffer, pH 7.4.

6) Sodium azide was added to the conjugated preparation (0.1%) which wasstored at 4° C.

EXAMPLE 5

An Ultra-sensitive Sandwich Two-step Back-titration TSH MicrospotImmunoassay employing Developing Antibody Conjugated to FluorescentMicrospheres

First Step

1) White polystyrene microtitre wells (Microlite 1 from DynatechLaboratories) were spotted with 1 μl or less of a 200 μg/ml monoclonalanti-TSH capture antibody in 0.1M phosphate buffer at pH 7.4. Theantibody droplets were aspirated immediately and the wells blocked with1% (w/v) BSA and washed extensively with the same buffer. The antibodymicrospots were kept in buffer until use.

2) After rinsing with 0.05M/l Tris-HCl buffer at pH 7.75 (wash buffer),200 μl of either standard in assay buffer or the sample was added toeach well and shaken for from 30 min to several hours at roomtemperature (or overnight at 4° C. if maximal assay sensitivity isdesired).

3) The wells were washed four times with wash buffer.

Second step

1) An aliquot of 200 μl of developing binding material antibodyconjugated to fluorescent-dye containing microspheres of diameter 0.1 μm(containing ˜0.01 mg antibody-conjugated microspheres) in assay bufferwas added to each well and shaken for 0.5 to 1 hour at room temperature.

2) The wells were washed seven times with the wash buffer whichcontained 0.05% Tween-20 (w/v), aspirated until completely dry andscanned with an MRC-600 Laser Scanning Confocal Microscope (Bio-RadMicroscience). The signal emitted from each antibody microspot wasquantified and the results were compared with the standard dose-responsecurve to determine TSH concentrations in unknown samples.

The standards used for production of the dose-response curve contained0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μU TSH/ml in the assay buffer.

The assay buffer composition was:

    ______________________________________                                        Tris-(hydroxymethyl)-aminomethane                                                                 50        mM/l                                            Sodium chloride     9.0       g/l                                             Bovine serum albumin                                                                              5.0       g/l                                             Bovine globulin     0.5       g/l                                             Tween 40            0.1       g/l                                             Sodium azide        0.5       g/l                                             HCl                 to a pH of 7.75 at 25° C.                          ______________________________________                                    

EXAMPLE 6

An assay for thyroid stimulating hormone (TSH) was carried out using twomonoclonal antibodies directed at different epitopes on the TSH moleculeas capture and developing antibodies, and TSH standard samples suppliedby the National Institute for Biological Standards and Control. Thecapture antibody was deposited as microspots approximately 0.5 mm indiameter on Dynatech Microlite microtitre wells by passive adsorption,giving a surface density of about 40,000 IgG molecules/μm². Thedeveloping antibody was covalently coupled to carboxylate-modifiedpolystyrene latex FluoSpheres 0.08 μm in diameter containingyellow/green fluorescent dye. The TSH samples were applied to themicrotitre wells in amounts of about 200 μl.

Following overnight incubation, the results obtained were as plotted onFIG. 6 of the accompanying drawings, which is a graph of fluorescenceintensity (y-axis) in arbitrary units against TSH concentration (x-axis)in mU/liter. The sensitivity of the assay (based on measurements of thestandard deviation of the zero dose estimate) was 0.002 mU/liter. Thesame standards and assay buffer as those used in Example 5 wereemployed.

EXAMPLE 7

Example 6 was repeated except that the total incubation time was reducedto 1 hour (0.5 hour incubation of sample with capture antibody, followedby 0.5 hour incubation with developing antibody) and the size of themicrospheres was increased to 0.12 μm diameter. The sensitivity of theassay was thereby increased ten-fold to 0.0002 mU/liter, based onmeasurements of the standard deviation of the zero dose estimate. Theresults are plotted in FIG. 7 of the accompany drawings, which is agraph on the same axes as FIG. 6.

EXAMPLE 8

A Single-step Ultra-sensitive Sandwich TSH Microspot Immunoassay UsingDeveloping Antibody Conjugated to Fluorescent Microspheres

1) White polystyrene microtitre wells (Microlite 1 from DynatechLaboratories) were spotted with 1 μl or less of a 200 μg/ml monoclonalanti-TSH capture antibody in 0.1M phosphate buffer at pH 7.4. Theantibody droplets were aspirated immediately and the wells blocked with1% (w/v) BSA and washed extensively with the same buffer. The antibodymicrospots were kept in buffer until use.

2) The wells were rinsed with the assay buffer used in Example 5, then100 μl of standard in assay buffer/sample and 100 μl of developingantibody-conjugated microspheres were added to each well and shaken atroom temperature for 30 minutes, or longer if maximal assay sensitivitywas desired.

3) The wells were washed seven times with wash buffer containing 0.05%Tween-20 (w/v), aspirated until completely dry and scanned with theconfocal microscope as in Example 5 above, the results being comparedwith the standard dose-response curve obtained using the standardsmentioned in Example 5 to determine the TSH concentration in unknownsamples.

EXAMPLE 9

Dual-labelled Ultra-sensitive Sandwich Single- or Two-stepBack-titration TSH Microspot Immunoassay Using Developing AntibodyConjugated to Fluorescent Microspheres

The protocols for the dual-labelled single or two-step assays areessentially the same as those for the single labelled assays describedabove except the unlabeled capture antibody is either labelled withTexas Red (Molecular Probes Inc.) and deposited directly on the whiteDynatech Microlite microtitre wells; or it can be coupled together withavidin to latex microspheres (Molecular Probes Inc) containing redfluorescent dye and the conjugated microspheres are then allowed to bindto a biotin-labelled BSA microspot deposited previously on themicrotitre wells.

EXAMPLE 10

A dual-label assay was carried out. The developing antibody wasconjugated to yellow/green polystyrene latex microspheres of 0.12 μmdiameter as described in Examples 1, 2 and 3. The capture antibody wasdeposited indirectly on Dynatech Microlite microtitre wells at a surfacedensity of about 40,000 IgG molecules/μm² via biotin/avidin. Theantibody was first conjugated together with avidin to polystyrene latexmicrospheres of 0.1 μm diameter containing red fluorescent dye, theconjugated spheres then being allowed to bind to biotinylated BSAmicrospots previously coated on the microtitre wells. The yellow/greenand orange/red dyes were scanned using the 488 and 568 nm lines of thekrypton/argon mixed-gas laser. This could be done either simultaneouslyor consecutively. The concentration of antigen (TSH) in the test samplewas obtained by observing the ratio of the fluorescent signals from thetwo dyes and correlating it with the signals using the standard samples.

The results obtained are shown in FIG. 8 of the accompanying drawings,which is a graph of the ratio of the two fluorescent signals (y-axis)against TSH concentration (x-axis) in mU/liter. The sensitivity of theassay (based on measurements of the standard deviation of the zero doseestimate) was 0.0002 mU/liter.

EXAMPLE 11

Single-labelled or Dual-labelled Ultra-sensitive Sandwich Single- orTwo-step Back-titration TSH Microspot Immunoassay Using BiotinylatedDeveloping antibody and a Universal Reagent of Avidin conjugatedFluorescent Microspheres

In contrast to the assay systems described in Examples 5 to 10, auniversal marker reagent of avidin conjugated fluorescent microsphereswas used in this Example to tag indirectly the bound developing antibodywhich had been labelled with biotin.

Although this assay system requires an additional step of the additionof avidin microspheres after the completion of the immunologicalreactions, the advantage of being able to use a "universal marker"outweighs this minor drawback. The "universal marker" system would beparticularly useful in a microspot multianalyte system described byRoger Ekins in WO-89/01157 because of the considerable improvements inassay sensitivity that can be expected as a result of the reduction innon-specific binding from employing a single universalavidin-microsphere preparation rather than the large number (equivalentto the number of simultaneous assays being performed) of developingantibody conjugated microsphere preparations that would otherwise berequired.

EXAMPLE 12 Microspot DNA Sequence Assay Methodologies

Non-competitive methodoloaies (qualitative and quantitative assays)

EXAMPLE 12a

Microspot sandwich DNA sequence assay using a biotinylated solid-phasedcapture DNA probe and anti-double-stranded DNA antibody conjugated tomicrospheres containing fluorescent dye.

1) Microlite 1 microtitre wells were spotted with 1 μl or less of 100μg/μl Avidin DX (Vector Laboratories) by adsorption for 1 hour at roomtemperature, blocked for 20 min with 200 μl of 0.1M Tris-HCl pH 7.5containing 0.15M NaCl, 0.05% Tween 20, 0.5% BSA and 100 μg/ml salmonsperm DNA and washed with Tris-HCl containing 0.05% Tween 20.

2) 5 to 100 ng of the biotinylated capture DNA probe purchased fromClontech Laboratories in 100 μl of Tris EDTA was added to the avidin DXcoated wells, incubated with shaking for 2 hr at room temperature andwashed with Tris-HCl containing 0.05% Tween 20.

3) In a modification of the procedure described in the Manero et alreference (see above) samples were prepared by boiling 0.5 ml aliquotsof the unknown samples for 10 min, then cooled rapidly on ice, dilutedwith hybridization buffer containing: 1X SSC (150 mmol/l of NaCl and 15mmol of trisodium citrate per liter), 2X Denhardt's solution (0.4 g ofBSA, 0.4 g of Ficol and 0.4 g of polyvinylpyrrolidone per liter), 10mmol/l Tris-HCl pH7.5, and 1 mmol/l EDTA. For a quantitative assay, theprepared samples or, as the case may be, standards containingsingle-stranded target DNA in known amounts and in the samehybridisation buffer were added to the wells (positive and negativecontrols rather than the standards were added to the other wells for thequalitative tests), incubated with shaking at 50° C. for 1 hour andwashed with PBS containing 0.05% Tween 20.

4) 200 μl of anti-double-stranded DNA antibody conjugated to fluorescentmicrospheres of diameter 0.1 μm (FluoSpheres) by the method described inExample 3 and in PBS containing 0.5% BSA and 0.05% Tween 20 was added,incubated with shaking for 1 hour at room temperature, washed withPBS-Tween 20 and scanned with the confocal microscope as described inExample 5.

EXAMPLE 12b

Microspot sandwich DNA sequence assay using a biotinylated solid-phasedcapture DNA probe, a complementary but non-overlapping developing DNAprobe labelled with digoxigenin and anti-digoxigenin antibody conjugatedto microspheres containing fluorescent dye.

1) The avidin-biotinylated capture DNA probe microspots were prepared asdescribed in Example 12a.

2) The hybridisation step was carried out as described in Example 12a.

3) 5 to 10 ng of the complementary but non-overlapping developing DNAprobe labelled with digoxigenin (as described in the Nickerson et alreference above) in 100 μl of hybridization buffer was added, incubatedwith shaking at 50° C. for 1 hour and washed with PBS-Tween 20.

4) 200 μl of the anti-digoxigenin antibody-conjugated fluorescentmicrospheres of about 0.1 μm diameter (FluoSpheres) in PBS containing0.5% BSA and 0.05% Tween 20 were added, incubated for 1 hour at roomtemperature with shaking, washed with PBS-Tween 20 and scanned with theconfocal microscope.

EXAMPLE 12c-12e

Microspot sandwich DNA sequence assay using an unlabelled solid-phasedcapture DNA probe and either anti-double-stranded DNA antibodyconjugated to microspheres containing fluorescent dye or a complementarybut non-overlapping biotinylated developing DNA developing probe andavidin-conjugated microspheres containing fluorescent dye or acomplementary but non-overlapping developing DNA probe labelled withdigoxigenin and antidigoxigenin antibody conjugated to microspherescontaining fluorescent dye may be carried out in a similar manner withappropriate changes.

Competitive DNA sequence assay methodology (quantitative andqualitative)

EXAMPLE 12f

Microspot DNA sequence assay using a biotinylated solid-phased captureDNA probe and a competitive material of target DNA sequence labelledwith microspheres containing fluorescent dye.

1) The avidin-biotinylated capture DNA probe microspots were prepared asdescribed in Example 12a.

2) Samples were prepared by boiling 0.5 ml aliquots of the unknownsamples for 10 min, then cooled rapidly on ice, and diluted withhybridization buffer containing: 1X SSC (150 mmol/l of NaCl and 15 mmolof trisodium citrate per liter), 2X Denhardt's solution (0.4 g of BSA,0.4 g of Ficol and 0.4 g of polyvinylpyrrolidone per liter), 10 mmol/lTris-HCl pH 7.5, and 1 mmol/l EDTA. For a quantitative assay, theprepared sample plus the competitive material of target DNA sequencesgenerated by polymerase chain reaction and labelled with fluorescentmicrospheres of diameter 0.1 μm (FluoSpheres) prepared using a techniquemodified from that described in the Wolf et al reference mentioned abovefor the attachment of DNA to latex particles, or standards containingsingle-stranded target DNA plus the competitive material and in the samehybridization buffer were added to the wells (positive and negativecontrols plus the competitive material instead of the standards plus thecompetitive material were added for the qualitative tests), incubatedwith shaking at 50° C. for 1 hour, washed with Tris-HCl containing 0.05%Tween 20 and scanned as described in Example 5.

As indicated above, these very high sensitivities for non-competitiveimmunoassays are unexpected in the light of the currently accepted viewson assay design. Some increase in sensitivity would be expected in anyassay format, once the idea of using microspheres in accordance with theinvention has been appreciated, because of the increased number ofmolecules of label attached to each molecule of developing bindingmaterial, this resulting in an effective increase in specific activityof the labelled developer molecules. However, this effect alone mightnot be expected to result in assay designs departing so markedly fromconventional concepts in this field and requiring in particular verysmall amounts of capture binding agent.

Two further possible explanations for these unexpected findings canperhaps be advanced. The first is that by confining a very small numberof capture binding agent molecules at high surface density to a verysmall area in the form of a microspot the signal/noise ratios obtainedin any finite incubation time may be improved as compared with thoseobtained in conventional designs in which very large amounts of captureantibody are distributed over large surface areas. The second is thatwhen analyte molecules are located between two solid surfaces on whichthe capture binding agent and developing binding material molecules arerespectively located (viz the microspheres and the microtitre wells inwhich the assay is performed) binding sites on the analyte molecules maybecome bound to multiple developing binding material molecules if theanalyte contains the same epitope replicated on its surface or if thedeveloping binding material is a polyclonal antibody or if more than onemonoclonal antibody directed at different epitopes on the analyte isused as developing material, thus increasing the effective affinity ofthe developing binding material. This implies that the surface densityof developing binding material molecules on the microspheres is likelyto represent an important determinant of the sensitivities achieved.

We claim:
 1. A binding assay process for determining the concentration of one or more analytes in a liquid sampleusing a capture binding agent having binding sites specific for each analyte expected to be present in the sample and a developing binding material capable of binding to bound analyte, to binding sites of the capture binding agent occupied by bound analyte or to binding sites remaining unoccupied by the analyte, the capture binding agent for a given analyte being immobilized at high density on a support in the form of one or more microspots each having an area less than 1 mm², and wherein labelled microspheres having a diameter less than 5 μm being used in the assay in relation to the developing binding material, so that the strength of the signal from the label is representative of the fractional occupancy of the binding sites of the capture binding agent, thereby allowing the concentration of the analyte to be determined.
 2. The process of claim 1, wherein the capture binding agent specific for a given analyte is used in a small amount that binds less than 5% of the analyte in the sample.
 3. The process of claim 2, wherein less than 0.1 V/K moles of a capture agent specific for a given analyte are used, where V is the sample volume in liters and K is the effective affinity constant of the capture binding agent for the analyte under the conditions of the assay.
 4. The process of claim 1, wherein the capture binding agent is immobilized at a surface density of 10,000 to 50,000 molecules/μm².
 5. The process of claim 1, wherein the microspots have an area less than 100 μm².
 6. The process of claim 1, wherein the concentration of a plurality of analytes is determined in the same operation using a plurality of different capture binding agents, each capture binding agent having binding sites specific for a given analyte in the sample.
 7. The process of claim 6, wherein one or more developing binding materials are used in the assay, the developing binding materials and the labelled microspheres being capable of binding to each other, so that the same label is used in relation to the developing binding materials, with the microspots containing different capture binding agent being distinguished apart by their location on the support.
 8. The process of claim 7, wherein one of the microspheres and the developing binding materials is conjugated to biotin and the other to avidin or streptavidin.
 9. The process of claim 1, wherein the label is contained within the microspheres.
 10. The process of claim 1, wherein the label is a fluorescent label.
 11. The process of claim 1, wherein the microspheres are blocked to minimize their non-specific interactions with other materials.
 12. The process of claim 1, wherein the capture binding agent and the developing binding material are antibodies.
 13. The process of claim 1, wherein the analyte is a nucleic acid sequence, the capture binding agent is an oligonucleotide sequence capable of binding the analyte and the developing binding material is oligonucleotide sequence or an antibody capable of hybridizing to the analyte.
 14. A binding assay process for detecting the presence of one or more target nucleic acid sequences in a liquid sampleusing one or more capture binding agents comprising an oligonucleotide sequence capable of hybridizing to a given target nucleic acid sequence and a developing binding material comprising an oligonucleotide sequence capable of hybridizing to the bound target nucleic acid sequence, the capture binding agent for a given target nucleic acid sequence being immobilized at high density in the form of one or more microspots each having an area less than 1 mm², and wherein labelled microspheres having a diameter less than 5 μm are used in the assay in relation to the developing binding material, so that the signal from the label indicates the presence of the target nucleic acid sequence, thereby allowing the presence of said one or more target nucleic acid sequences to be detected.
 15. A kit for determining the concentration of one or more analytes in a liquid sample, the kit comprising:one or more capture binding agents, each capture binding agent having binding sites specific for a given analyte expected to be present in the sample, wherein the capture binding agents are immobilized at high density on a support in the form of one or more microspots, each microspot having an area less than 1 mm² ; and, one or more developing binding materials, each developing binding material being capable of binding to a given bound analyte, to binding sites of a given capture binding agent occupied by bound analyte or to binding sites of a given capture binding agent remaining unoccupied by the analyte; wherein labelled microspheres having a diameter less than 5 μm are used in the assay in relation to the developing binding material, so that the strength of the signal from the label is representative of the fractional occupancy of the binding sites of a given capture binding agent, thereby allowing the concentration of the analyte to be determined.
 16. The kit of claim 15, further comprising standards containing known amounts or concentrations of analyte. 